Abstract Background and Aims Nephronophthisis (NPH) is an autosomal recessive cystic kidney disease, which is the most common genetic cause of end-stage renal disease (ESRD) in children. The pathogenesis of NPH has not been clear yet. There are more than 20 different pathogenic genes identified at present, among which NPHP1 is the most prevalent one. Hippo signal pathway has been suggested to be involved in the pathogenesis of NPH cause by NPHP4 and NPHP9 defects, respectively. In the present study, we attempted to explore the state of hippo signal pathway in NPHP1 defect in both MDCK cells and C57/6j mice. Method NPHP1 knockout (NPHP1KO) MDCK cells and NPHP1KO C57BL/6J mice were constructed by CRISPR/Cas9 technology. Recombined kibra knockdown siRNA, kibra knockdown AAV9 (AAV9-Kibra-KD) and NPHP1 overexpress AAV9 construct (AAV9-NPHP1-OE) were constructed, respectively. The expression and phosphorylation of core molecules of hippo pathway in different group of MDCK cells and the kidney C57BL/6J mice were investigated through Real-time Quantitative PCR, western blotting, immunofluorescence. The renal pathology of the mice kidney was also observed. Results In NPHP1KO MDCK cells and the kidney of NPHP1KO mice, the expression of KIBRA, p-MST, p-LATS and p-Yap were increased, which indicates the activation of hippo signal pathway. The activation of hippo pathway could be suppressed partially by NPHP1 re-expression both in NPHP1KO MDCK cells and the kidney of NPHP1KO mice, which means the activation of hippo pathway was related to NPHP1 defect both in vitro and in vivo. Moreover, the kidney pathological changes were improved by NPHP1 re-expression. Kibra is the upper regulator of hippo signal pathway, we observed that kibra knockdown inhibited the activation of hippo pathway both in NPHP1KO MDCK cells and the kidney of NPHP1KO mice, which indicates that kibra mediates the activation of hippo pathway related to NPHP1 defect both in vitro and in vivo. As well, kibra knockdown ameliorated renal fibrosis CTGF expression, interstitial inflammation and renal cyst formation in NPHP1KO mice. Conclusion We conclude that NPHP1 defect activates hippo pathway which involves in the pathogenesis of NPH. Kibra mediates the activation of hippo related to NPHP1 defect by classic signal pathway. Hippo signal maybe a target for the treatment of NPHP1 defect NPH.